1. Field of the Invention
The invention relates to a monoclonal antibody which recognizes a glycosylated antigenic structure present on the hemagglutinin of both the type A flu virus and the type B flu virus, and also to the use thereof for diagnostic, therapeutic, and purification purposes.
2. Summary of the Related Art
There are 3 types of flu virus (types A, B and C) responsible for infectious pathologies in humans and animals. Currently, the type A and type B viruses are the agents responsible for the flu epidemics and pandemics observed in humans.
The type A viruses circulating mainly in humans and birds are subdivided into subtypes according to the antigenic structure of hemagglutinin (HA) and of neuraminidase (NA), which are the main constituents of the viral envelope. 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) have been identified. The subtype of a type A virus is defined by the HA subtype and the NA subtype that it carries. The H1N1 and H3N2 virus subtypes are those which currently circulate in humans. It is feared that the H5N1 subtype or the H7N1 subtype which circulate in birds will adapt to humans and be responsible for a new pandemic. It is not out of the question, either, that the H2N2 subtype which circulated between 1957 and 1968 in humans might reappear and also be responsible for a pandemic in individuals below the age of 40.
The type B virus strains are strictly human. The antigenic variations in the HA within the type B strains are weaker than those observed within the type A strains. The type B strains, isolated since the 1970s, are divided into two distinct phylogenetic and antigenic groups according to the genetic sequence of the HA (Lindstrom SE, Journal of Virology, 1999, pp 4413-4426). The B/Victoria/2/87 (B/Victoria) is the leading strain of the first group, while the B/Yamagata/16/88 (B/Yagamata) is the leading strain of the second group. The type B virus strains which were isolated in the 1980s belong especially to the B/Victoria group, whereas the strains which were isolated in the 1990s belong especially to the B/Yamagata group. In 1994, the B/Victoria group reemerged in China and, since then, type B strains belonging to the two groups have been isolated during the same flu season (Nakagawa N et al., Journal of Medical Virology, 2001, pp 745-750).
Flu virus HA is, in its natural form, a trimeric glycoprotein with a molecular weight of approximately 220,000 daltons. It is subjected to a strong “selection pressure” with, as a result, the appearance:                of major variations in its structure, resulting in an “antigenic shift”. These phenomena are observed in the type A viruses and are responsible for the appearance of reassortant viruses having a new HA subtype. These reassortant viruses may be responsible for new pandemics in humans or animals;        of minor variations in its structure due mainly to point mutations resulting in an “antigenic drift”. These phenomena are observed both in type A viruses and in type B viruses. They are responsible for the appearance of new viral strains which can trigger epidemics or sporadic flu foci during inter-pandemic periods.        
Flu virus HA also contains numerous glycosylation sites (between 7 and 9 on the H1 and H3 subtypes). The glycosylated chains are linked to the protein structure by means of N-glycosidic linkages which link an asparagine of the protein sequence to an N-acetylglucosamine of the sugar chain. Two types of glycosylated chains are conventionally distinguished according to their sugar composition: type I glycosylated chains contain essentially N-acetylglucosamine, mannose, galactose and fucose, and type II glycosylated chains contain essentially mannose and N-acetylglucosamine. The N-glycosylation of HA is carried out through the dolichol pathway that results in the transfer in block of the oligosaccharide sequence (glucose)3-(mannose)9-(N-acetylglucosamine)2 onto the asparagines which are in a consensus sequence of Asn-X-Ser/Thr type in the protein chain (Virology, 133, 77-91 (1984)). The oligosaccharide sequence transferred in block has a “three-antenna” configuration represented schematically in FIG. 2.
As was shown by Mir-Shekari S. Y. et al. (Journal of Biological Chemistry, 272, 4027-4036 (1997)), the initial three-antenna sequences transferred then individually undergo a transformation process according to their anchoring site on the HA protein chain. The glycosylated structures may finally be two-antenna, three-antenna or four-antenna structures, with a smaller or larger number of sugars on each of the antennae.
In order to prevent flu epidemics, vaccines containing two strains of virus A belonging to different subtypes (currently the H1N1 and H3N2 subtypes) and a type B virus strain are annually prepared. The vaccinal strains used take into account the variations which have occurred from one year to the other at the level of the HA protein sequence and which are responsible for the appearance of new epidemic foci. These new strains are identified and characterized using very precise diagnostic tools, in particular by means of monoclonal antibodies which reveal very specifically the variations observed. These monoclonal antibodies have a very narrow specificity since they generally recognize the HA of a single viral strain. They are “strain” specific.
Monoclonal antibodies having a broader specificity (referred to as “subtype” specificity also exist for characterizing type A viruses. They are “subtype” specific insofar as they recognize the HAs of viral strains having the same HA subtype but not the HAs of viral strains of another subtype. Monoclonal antibodies specific for the H1 subtype or for the H3 subtype in particular exist. A cross-reactive monoclonal antibody which recognizes both the HAs of flu viruses belonging to the H1N1 and H2N2 subtypes has been reported in U.S. Pat. No. 5,589,174.
As regards the monoclonal antibodies directed against the HA of type B viruses, N. Nakagawa has described a battery of monoclonal antibodies directed against the HA of strains belonging to the two groups B/Victoria and B/Yagamata. The monoclonal antibodies 10B8, 8E6, 1H12, 2H12 and 9E10 recognize the HA of type B virus strains belonging to the B/Victoria group which were isolated between 1996 and 1997, but not the HA of type B virus strains isolated during this same period and which belong to the B/Yamagata group (Nakagawa N et al., Journal of Virological methods (1999), 113-120). These monoclonal antibodies were obtained by immunizing mice with the B/Nagasaki/1/87 strain which belongs to the B/Victoria group. Monoclonal antibodies 1B2, 5B1, 5H4, 7H11, 8B3 and 9G6, obtained after immunization of mice with the B/Mie/1/93 strain (which belongs to the B/Yagamata group), which recognize the HA of type B virus strains belonging to the B/Yamagata group, have also been described (Nakagawa N et al., Journal of Medical Virology (2001), 745-750).